Vibrational Fingerprint Analysis of an Azo-based Resonance Raman Scattering Probe for Imaging Proton Distribution in Cellular Lysosomes

Abstract

Due to the fundamental mechanism of vibrational state transitions for chemical bonds, the spectra of Raman scattering are narrowbanded and photostable signals capable of probing specific reactions. In the case of protonation/deprotonation reactions, certain chemical bonds are broken and new bonds are formed. Based on the changes of the vibrational modes for the corresponding bonds, fingerprint analysis of multiple Raman bands may allow for the in situ visualization of proton distribution in live cells. However, Raman scattering faces the well-known challenge of low sensitivity. To perform the vibrational fingerprint analysis of Raman scattering by overcoming this challenge, we developed an azo-based resonance Raman pH probe. It was an azobenzene-featured small molecule responsive to protons with the inherent Raman signal ∼10 4 -fold more intense than that of the conventional alkyne-type Raman reporter 5-ethynyl-2′-deoxyuridine. Through the substitution of the electron-donating and -withdrawing entities to the azobenzene group, the effect of resonance Raman scattering and fluorescence quenching was obtained. This effect resulted in a significant Raman enhancement factor of ∼10 3 compared to the counterpart molecules without the molecular design. Based on the enhanced Raman sensitivity of the azo-based resonance Raman pH probe, the identification of vibrational fingerprint changes at the azo group was achieved during the protonation/deprotonation reactions, and the vibrational fingerprint analysis resolved a pH difference of less than 0.2 unit. The method enabled sensitive hyperspectral cell imaging that clearly visualized the change of proton distribution in autophagic cells.

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes

“…In comparison, advanced coherent Raman techniques require >100-fold higher laser powers to achieve similar speeds using previously reported probes and must repeat the scan for each additional spectral channel (see Table S2 in the Supporting Information). Furthermore, DAERI’s full-spectrum information would be significant for detecting subtle spectral variations caused by local environment changes, for example, the local pH changes. , …”

“…Furthermore, DAERI's full-spectrum information would be significant for detecting subtle spectral variations caused by local environment changes, for example, the local pH changes. 16,17 High-Speed DAERI of Subcellular Organelle Dynamics. Mitochondrial and lysosomal dynamic interactions are of intense research interest because of their significant role in cellular metabolism as well as in cancer biology 31,32 In approximately 3 min, we observed two connected lysosomes separating from each other (Figure 3b, white circle).…”

“…In addition, with the spatiotemporal resolution and SNR high enough to clearly visualize, quantify and characterize organelle function and dynamics within living cells, a potential attractive aspect of DAERI is to provide functional information on the local cellular environment, 17 which remains a cutting-edge target for future studies. Local environmental changes such as pH leads to subtle differences in cellular Raman spectrum, such that the full hyperspectral information of DAERI may offer a distinct advantage over single-plex or few-plex techniques.…”

“…Small-molecule Raman probes present narrow spectral bands reflecting their vibrational fingerprints and provide an attractive solution to enable dynamic Raman imaging. They are generally cell-permeable, target-specific, and with low toxicity for biomolecular imaging in living cells. − Based on the full-spectrum information, Raman probes feature greater ability for multiplex imaging that indicates more specific responses to the local chemical environment. − Since the first demonstration of Raman cellular imaging based upon 5-ethynyl-2′-deoxyuridine (EdU), many alkyne-tagged Raman probes have been developed as bio-orthogonal reporters in the cellular Raman-silent region (1800–2800 cm –1 ). ,− Additionally, some resonance Raman probes enhance the sensitivity of spontaneous Raman imaging , and stimulated Raman imaging ,− by coupling electronic and vibrational transitions. However, these Raman probes are not sensitive enough to perform high-resolution imaging of fast cellular dynamics without the requirement of coherent optics and high excitation power.…”

Correlating the orbital overlap area and vibrational frequency shift of an isocyanide moiety adsorbed on Pt and Pd covered Au(111) surfaces

“…To sensitively detect the electronic properties of metal surfaces experimentally, molecules containing an isocyanide moiety, for instance phenyl isocyanide (PIC), are often used as a probe in vibrational spectroscopy. [30][31][32][33][34][35][36][37] This is because the isocyanide moiety has a unique CRN stretching (n CRN ) vibrational peak in the 2000-2500 cm À1 region, in which there is scarcely any other vibrational modes appearing, except for the alkyne moiety and carbon monoxide (CO). [38][39][40][41][42] More importantly, the vibration of the isocyanide moiety can be easily influenced by the change of the geometric and electronic structures of the metal surfaces onto which the molecules adsorb, due to their strong orbital interactions.…”